Kei1: A Novel Subunit of Inositolphosphorylceramide Synthase, Essential for Its Enzyme Activity and Golgi Localization

Sato, Keisuke; Noda, Yoichi; Yoda, Koji

doi:10.1091/mbc.e09-03-0235

Cited by 50 publications

(36 citation statements)

References 59 publications

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“…Absolute colony numbers for cell viability assays are in Supplementary Table 1. kei1-1 csg2Δ cells were assayed at 30°C, the semi-permissive temperature. 18 Mean values from n ≥ 3 with error bars indicating S.E.M. (c) Simplified diagram of sphingolipid synthesis pathway including major intermediates, and relevant proteins catalyzing specific steps.…”

Section: Resultsmentioning

confidence: 99%

Sphingolipid accumulation causes mitochondrial dysregulation and cell death

et al. 2017

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Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2Δ mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In these cells, aberrant activation of Ras GTPase is IPC-dependent, and accompanied by increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial mass. Survival or death of csg2Δ cells depends on nutritional status. Abnormal Ras activation in csg2Δ cells is associated with impaired Snf1/AMPK protein kinase, a key regulator of energy homeostasis. csg2Δ cells are rescued from ROS production and death by overexpression of mitochondrial catalase Cta1, abrogation of Ras hyperactivity or genetic activation of Snf1/AMPK. These results suggest that sphingolipid dysregulation compromises metabolic integrity via Ras and Snf1/AMPK pathways.Sphingolipids are critical structural molecules in cell membranes, forming membrane microdomains by associating with cholesterol and specific proteins.1 Sphingolipid metabolites are also important signaling molecules linked to multiple other metabolic pathways with kinases and phosphatases as regulatory targets.2,3 Sphingolipids have roles in numerous cell processes, including regulation of mitochondrial function, cell death and aging.2,4 Cellular sphingolipid homeostasis is maintained by control of synthesis, breakdown and interorganellar transport of sphingolipid metabolites.1 The importance of sphingolipids is underscored by several lysosomal storage disorders, including Tay Sachs, Gaucher and Nieman-Pick diseases, which are attributable to defective sphingolipid breakdown; similarly, a hereditary sensory neuropathy is caused by accumulation of abnormal sphingolipid metabolites.

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Section: Resultsmentioning

confidence: 99%

Sphingolipid accumulation causes mitochondrial dysregulation and cell death

et al. 2017

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“…Tagging of Kre6 with three copies of the HA epitope at their C termini was done by homologous recombination between an appropriate fragment on the plasmid and the chromosome as described previously (13). The expression units of N-terminal-truncated Kre6 derivatives were constructed by joining the PCR products of the 300-bp 5Ј-upstream region of KRE6, truncated KRE6 ORFs with extraneous start codon and BamHI site, and 3HA-TDH1 terminator , and these were integrated at the chromosomal ura3-52 locus of KTY604 (kre6⌬::kanMX4).…”

Section: Methodsmentioning

confidence: 99%

Kre6 Protein Essential for Yeast Cell Wall β-1,6-Glucan Synthesis Accumulates at Sites of Polarized Growth

Kurita

Noda

Такаги

et al. 2011

Journal of Biological Chemistry

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Saccharomyces cerevisiae Kre6 is a type II membrane protein with amino acid sequence homology with glycoside hydrolase and is essential for ␤-1,6-glucan synthesis as revealed by the mutant phenotype, but its biochemical function is still unknown. The localization of Kre6, determined by epitope tagging, is a matter of debate. We raised anti-Kre6 rabbit antiserum and examined the localization of Kre6 and its tagged protein by immunofluorescence microscopy, subcellular fractionation in sucrose density gradients, and immunoelectron microscopy. Integration of the results indicates that the majority of Kre6 is in the endoplasmic reticulum; however, a small but significant portion is also present in the secretory vesiclelike compartments and plasma membrane. Kre6 in the latter compartments is observed as strong signals that accumulate at the sites of polarized growth by immunofluorescence. The truncated Kre6 without the N-terminal 230-amino acid cytoplasmic region did not show this polarized accumulation and had a severe defect in ␤-1,6-glucan synthesis. This is the first evidence of a ␤-1,6-glucan-related protein showing the polarized membrane localization that correlates with its biological function.

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“…After incubation with peroxidase-conjugated secondary antibodies (KPL), detection was performed using enhanced chemiluminescence (Thermo Scientific). Indirect Immunofluorescence-The localizations of HAtagged mannosyltransferases and Mnn9 were observed by indirect immunofluorescence as described previously (19). Anti-HA mouse monoclonal antibody or rabbit anti-Mnn9 antibodies diluted to 1:50 were used as primary antibodies.…”

Section: Methodsmentioning

confidence: 99%

Svp26 Facilitates Endoplasmic Reticulum to Golgi Transport of a Set of Mannosyltransferases in Saccharomyces cerevisiae

Noda

Yoda

2010

Journal of Biological Chemistry

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Svp26 is a polytopic integral membrane protein found in the ER and early Golgi compartment. In the ⌬svp26 cell, the Golgi mannosyltransferase Ktr3 remains in the ER. Here, we report that two other Golgi mannosyltransferases, Mnn2 and Mnn5 are also mislocalized and found in the ER in the absence of Svp26 and that localization of other mannosyltransferases including Mnn1 are not affected. Mnn2 and Mnn5 bind to Svp26 in vivo as Ktr3 does. Using an in vitro budding assay, the incorporation of Ktr3 and Mnn2 in the COPII vesicles is greatly stimulated by the presence of Svp26. As Svp26 itself is an efficient cargo, Svp26 is likely to support selective incorporation of a set of mannosyltransferases into COPII vesicles by working as their adaptor protein. The domain switching between Svp26-dependent Mnn2 or Ktr3 and Svp26-independent Mnn1 suggests that the lumenal domain of mannosyltransferases, but not the cytoplasmic or transmembrane domain, is responsible for recognition by Svp26.

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Kei1: A Novel Subunit of Inositolphosphorylceramide Synthase, Essential for Its Enzyme Activity and Golgi Localization

Cited by 50 publications

References 59 publications

Sphingolipid accumulation causes mitochondrial dysregulation and cell death

Sphingolipid accumulation causes mitochondrial dysregulation and cell death

Kre6 Protein Essential for Yeast Cell Wall β-1,6-Glucan Synthesis Accumulates at Sites of Polarized Growth

Svp26 Facilitates Endoplasmic Reticulum to Golgi Transport of a Set of Mannosyltransferases in Saccharomyces cerevisiae

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